Methods for detection of hydrophobic drugs

ABSTRACT

Methods and reagents are disclosed for pretreating a sample suspected of containing a hydrophobic drug for conducting an assay method for detecting the hydrophobic drug. A combination is provided in a medium. The combination comprises (i) the sample, (ii) a releasing agent for releasing the hydrophobic drug and the metabolites from endogenous binding moieties, and (iii) a selective solubility agent that provides for substantially equal solubility of the hydrophobic drug and the metabolites in the medium. The selective solubility agent comprises a water miscible, non-volatile organic solvent and is present in the medium in a concentration sufficient to provide for substantially equal solubility of the hydrophobic drug and the metabolites in the medium. The medium, which may further comprise a hemolytic agent, is incubated under conditions for releasing the hydrophobic drug and the metabolites from endogenous binding moieties. For conducting an assay for the hydrophobic drug, the above pretreatment is performed and to the medium is added reagents for determining the presence and/or amount of the hydrophobic drug in the sample wherein the reagents comprise at least one antibody for the hydrophobic drug. The medium is examined for the presence of a complex comprising the hydrophobic drug and the antibody for the hydrophobic drug, the presence and/or amount of the complex indicating the presence and/or amount of the hydrophobic drug in the sample.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Divisional of U.S. patent application Ser. No.11/956,603 filed on Dec. 14, 2007, now U.S. Pat. No. 7,910,378 issuedMar. 22, 2011.

BACKGROUND

The invention relates to compounds, methods and kits for thedetermination of hydrophobic drugs such as, for example,immunosuppressant drugs, in samples, such as patient samples, known orsuspected to contain one or more of such hydrophobic drugs.

The body relies upon a complex immune response system to distinguishself from non-self. At times, the body's immune system must becontrolled in order to either augment a deficient response or suppressan excessive response. For example, when organs such as kidney, heart,heart-lung, bone marrow and liver are transplanted in humans, the bodywill often reject the transplanted tissue by a process referred to asallograft rejection.

In treating allograft rejection, the immune system is frequentlysuppressed in a controlled manner with drug therapy. Immunosuppressantdrugs are carefully administered to transplant recipients in order tohelp prevent allograft rejection of non-self tissue. Two most commonlyadministered immunosuppressive drugs to prevent organ rejection intransplant patients are Cyclosporine (CSA) and FK-506 (FK ortacrolimus). Another drug that finds use as an immunosuppressant in theUnited States and other countries is sirolimus, also known as rapamycin.Derivatives of sirolimus are also said to be useful asimmunosuppressants. Such derivatives include, for example, Everolimus,and the like.

The side effects associated with some immunosuppressant drugs can becontrolled in part by carefully controlling the level of the drugpresent in a patient. Therapeutic monitoring of concentrations ofimmunosuppressant drugs and related drugs in blood is required tooptimize dosing regimes to ensure maximal immunosuppression with minimaltoxicity. Although immunosuppressant drugs are highly effectiveimmunosuppressive agents, their use must be carefully managed becausethe effective dose range is often narrow and excessive dosage can resultin serious side effects. On the other hand, too little dosage of animmunosuppressant can lead to tissue rejection. Because the distributionand metabolism of an immunosuppressant drug can vary greatly betweenpatients and because of the wide range and severity of adversereactions, accurate monitoring of the drug level is essential.

In therapeutic drug monitoring field, selectively detecting the parentdrug over its metabolites is often an important goal for designingimmunoassays. This is especially true for immunosuppressant drugs. Forthat reason, HPLC tandem MS assays have become standard methods for themeasurement of sirolimus and other immunosuppressant drugs due to theirability to selectively measure the parent drug.

Most whole blood assays for immunosuppressant drugs require a manualstep using reagents to extract the drug from blood constituents. As aresult, the drug molecules and drug metabolite molecules are dissociatedfrom endogenous binding proteins and are extracted into a relativelyclean solution in which plasma proteins and lipoprotein particles aswell as most other molecules are removed. Because precipitationtechniques are usually used, the extracted sample is basically free ofmost blood macromolecules including drug-binding proteins. Thus, in theextracted samples, the parent drug and its metabolites are dissolved asunbound, individual molecules and compete with one another for reactionwith an assay antibody in the immunoreaction mixture. The binding ofassay antibody to the drug occurs in the absence of most endogenoussubstances in these assays. The cross-reactivity of a drug metabolitedepends mostly on its antibody binding affinity in such assays.

In a homogeneous assay for an immunosuppressant drug where there is nomanual extraction or separation of the drug from blood constituents, anantibody for the immunosuppressant drug has to detect the drug in thepresence of most or all blood constituents, the presence of which mightinterfere with the binding of the antibody to the immunosuppressantdrug. Furthermore, the samples contain metabolites of the drug and highmetabolite cross-reactivity presents a serious accuracy issue in assaysfor immunosuppressant drugs.

There is, therefore, a continuing need to develop fast and accuratediagnostic methods to measure levels of immunosuppressant drugs orderivatives thereof in patients. The methods should be fully automatedand be accurate even when conducted on whole blood samples withno-extraction using a homogeneous assay where an antibody employed inthe assay has to detect the drug in the presence of most, if not all,blood constituents and in the presence of drug metabolites. The assayshould selectively detect the parent drug while minimizing inaccuraciesresulting from the cross-reactivity of its metabolites.

SUMMARY

One embodiment of the present invention is a method for selectivelyenhancing the bioavailability of a hydrophobic drug over metabolites ofthe hydrophobic drug. A combination is provided in a medium. Thecombination comprises (i) the sample, (ii) a releasing agent forreleasing the hydrophobic drug and its metabolites from endogenousbinding moieties, and (iii) a selective solubility agent that providesfor enhancement of the bioavailability of the hydrophobic drug over thatof the metabolites in the medium. The selective solubility agentcomprises a water miscible, non-volatile organic solvent and is presentin the medium in a concentration sufficient to enhance thebioavailability of the hydrophobic drug over that of the metabolites inthe medium. The medium is incubated under conditions for enhancing thebioavailability of the hydrophobic drug over that of the metabolites.

Another embodiment of the present invention is a method for determininga hydrophobic drug in a sample suspected of containing a hydrophobicdrug. A combination is provided in a medium. The combination comprisesthe sample, a releasing agent for releasing the hydrophobic drug and itsmetabolites from endogenous binding moieties, and a selective solubilityagent that provides for enhancement of the bioavailability of thehydrophobic drug over that of the metabolites in the medium, wherein theselective solubility agent comprises a water miscible, non-volatileorganic solvent and wherein the concentration of the selectivesolubility agent in the medium is sufficient to enhance thebioavailability of the hydrophobic drug over that of the metabolites inthe medium. The combination in the medium further comprises a hemolyticagent. The medium is incubated under conditions for hemolyzing cells inthe sample and for enhancing the bioavailability of the hydrophobic drugover that of the metabolites. To the medium is added reagents fordetermining the presence and/or amount of the hydrophobic drug in thesample wherein the reagents comprise at least one antibody for thehydrophobic drug. The medium is examined for the presence of a complexcomprising the hydrophobic drug and the antibody for the hydrophobicdrug, the presence and/or amount of the complex indicating the presenceand/or amount of the hydrophobic drug in the sample.

Another embodiment of the present invention is a method for determiningan immunosuppressant drug in a sample suspected of containing animmunosuppressant drug. A combination is formed in a medium wherein thecombination comprises the sample, a releasing agent for releasing theimmunosuppressant drug and its metabolites from endogenous bindingmoieties and a selective solubility agent that provides for enhancementof the bioavailability of the hydrophobic drug over that of themetabolites in the medium. The selective solubility agent comprises awater miscible, non-volatile organic solvent. The concentration of theselective solubility agent in the medium is sufficient to provide forenhancement of the bioavailability of the hydrophobic drug over that ofthe metabolites in the medium. The medium is incubated under conditionsfor releasing the immunosuppressant drug and its metabolites fromendogenous binding moieties. To the medium is added (i) a reagentcomprising (I) an antibody for the immunosuppressant drug and (II) anenzyme and (ii) magnetic particles comprising the immunosuppressant drugor an analog thereof. The medium is examined for the presence of acomplex comprising the immunosuppressant drug and the antibody for theimmunosuppressant drug, the presence and/or amount of the complexindicating the presence and/or amount of the immunosuppressant drug inthe sample.

Another embodiment of the present invention is a method for determiningan immunosuppressant drug in a sample suspected of containing animmunosuppressant drug. A combination is formed in a medium wherein thecombination comprises the sample, a releasing agent for releasing theimmunosuppressant drug and its metabolites from endogenous bindingmoieties and a selective solubility agent that provides for enhancementof the bioavailability of the hydrophobic drug over that of themetabolites in the medium. The selective solubility agent comprises awater miscible, non-volatile organic solvent and the concentration ofthe selective solubility agent in the medium is sufficient to providefor enhancement of the bioavailability of the hydrophobic drug over thatof the metabolites in the medium. The medium is incubated underconditions for enhancing the bioavailability of the hydrophobic drugover that of the metabolites. To the medium is added (i) aphotosensitizer associated with a first particle and being capable ofgenerating singlet oxygen, and (ii) a chemiluminescent compositionactivatable by singlet oxygen and associated with a second particle,wherein an antibody for the immunosuppressant drug is associated withthe first particle or the second particle or both. The combination issubjected to conditions for binding of the antibody to theimmunosuppressant drug, if present. The photosensitizer is irradiatedwith light and the amount of luminescence generated by thechemiluminescent composition is detected. The amount of luminescence isrelated to the amount of the immunosuppressant drug in the sample.

Alternatively, in the above embodiment, one of the first particle or thesecond particle comprises the antibody and the other particle comprisesa drug analog for the immunosuppressant drug. The combination issubjected to conditions for competition of the drug analog coatedparticles and the immunosuppressant drug, if present, to the antibodyfor the drug. Alternatively, in the above embodiment, the first particleor the second particle comprises streptavidin, which combines with abiotinylated analog for the immunosuppressant drug in the medium. Thecombination is subjected to conditions for competition of biotinylateddrug analog and the immunosuppressant drug for the antibody for thedrug. In either of the above alternative embodiments, thephotosensitizer is irradiated with light and the amount of luminescencegenerated by the chemiluminescent composition is detected. The amount ofluminescence is related to the amount of the immunosuppressant drug inthe sample.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

General Discussion

The present inventors have recognized that the cross-reactivity of drugmetabolites can be reduced by making the drug and metabolites lessprotein-bound and enhancing the solubility of the hydrophobic drug in anassay mixture relative to metabolites of the hydrophobic drug, which thepresent inventors recognized as more hydrophilic than the drug itself.The more hydrophilic nature of the drug metabolites appears to be due toextra hydroxyl groups that result from drug metabolism through the liverby demethylation and hydroxylation. Hydrophobic interaction is animportant and common mechanism for drug-protein binding in aqueousblood. The present inventors observed that hydrophilic metabolites tendto have lower affinity to the binding proteins and diffuse more freelyin aqueous blood and assay mixture. For that reason a larger portion ofthe metabolite molecules are free, non-protein bound and more accessibleto the assay antibody than the parent drug in the aqueous assay mixture.Assuming that a drug metabolite has the same binding affinity to theassay antibody as the parent drug, the metabolite will form moreimmuno-complexes with the antibody than the parent drug due to itshigher accessibility. The above recognition is contrary to a commonbelief that metabolite cross-reactivity is only a function of antibodybinding affinity. The present inventors have determined that thecross-reactivity of a metabolite in such assays depends not only on itsantibody binding affinity but also on its binding affinity to theendogenous binding proteins.

Embodiments of the assays described herein are homogeneous immunoassays,which may also be referred to as essentially partition-freeimmunoassays. Embodiments of the present assays selectively detect theparent drug while minimizing the cross-reactivity of an antibody for thedrug to the metabolites of such drug. The use of selective solubilityagents that are water miscible, non-volatile organic solvents in asample partition-free assay selectively increases the bioavailability ofthe parent drug over that of the metabolites, and selectively increasesthe accessibility of the hydrophobic drug to the assay antibody over themetabolites. The solubility differentials rearranged by the aboveselective solubility agents over regular aqueous reagent solutionsminimizes the detection of more hydrophilic metabolites and enhances thedetection of the parent hydrophobic drug. That is, the selectivesolubility agents of the invention selectively increase thebioavailability of the parent drug over the metabolites. Thus, theselective solubility agents added to an assay medium adjust theselectivity or bioavailability of the hydrophobic drug relative toaqueous media that are customarily employed in such assays. Theselective solubility agents may also enhance the bioavailability of ahydrophobic antibody that is employed in an assay.

The current methods focus on the mitigation of inaccurate assay resultscaused by cross-reactivity of drug metabolites with the antibody reagentemployed in an immunoassay. The present methods have application tofully automated homogeneous assays in which, prior to the assay, thereis no extraction or separation of the hydrophobic drug from otherconstituents of the sample including drug metabolites. In a “non-manualextraction” assay, a sample such as a whole blood sample is combinedwith a hemolyzing agent and a releasing agent in a medium and, followingan incubation period to allow for hemolysis and release of the drug fromother blood constituents, reagents for conducting an assay for thehydrophobic drug are added to the medium and the assay is conducted. Ithas been found that the bioavailability of a hydrophobic drug in anassay for the drug may be enhanced relative to the metabolites of thedrug by incubating a sample suspected of containing the hydrophobic drugwith a releasing agent and a selective solubility agent that enhancesthe availability of the hydrophobic drug for subsequent binding to anantibody for the drug during an assay to detect the presence and/oramount of the drug wherein other constituents of the sample are present.

The term “hydrophobic drug” as used herein refers to a drug, usually atherapeutic drug, where the drug exhibits a characteristic of absorptionby a lipophilic moiety such as, for example, a lipoprotein, or ofreduced solubility in a polar medium. The absorption or lack ofsolubility is such that it interferes with the quantitation of the drugin an assay for the drug. Interference with the quantitation of the drugmeans that the ability to make an accurate quantitative determination ofthe drug in an assay is reduced by at least about 10%, by at least about15%, by at least about 20%, by at least about 25%, by at least about30%, and so forth. A “hydrophobic antibody” is an antibody that exhibitsreduced solubility in an aqueous medium as compared to other antibodies.

Immunosuppressant drugs are an example of hydrophobic drugs.Immunosuppressant drugs are therapeutic drugs that are administered totransplant recipients in order to help prevent allograft rejection ofnon-self tissue. Immunosuppressive drugs can be classified as follows:glucocorticoids, cytostatics, antibodies, drugs acting on immunophilins,and other drugs such as interferons, opiates INF binding proteins,mycophenolate, FTY720 and the like. A particular class ofimmunosuppressant drugs comprises those drugs that act on immunophilins.Immunophilins are an example of high-affinity, specific binding proteinshaving physiological significance Two distinct families of immunophilinsare presently known: cyclophilins and macrophilins, the latter of whichspecifically bind, for example, tacrolimus or sirolimus. Theimmunosuppressant drugs that act on immunophilin include, for example,cyclosporin (including cyclosporin A, cyclosporin B, cyclosporin C,cyclosporin D, cyclosporin E, cyclosporin F, cyclosporin G, cyclosporinH, cyclosporin I), tacrolimus (FK506, PROGRAF®), sirolimus (rapamycin,RAPAMUNE®), everolimus (RAD, CERTICAN®) and so forth.

The term “bioavailability” as used herein with respect to a “hydrophobicdrug” refers to the amount of hydrophobic drug in a sample that isavailable for measurement such as, for example, available for binding toan antibody for the hydrophobic drug particularly in an assay wherethere are constituents in the sample to be analyzed such as metabolitesof the drug that cross-react with an antibody for the drug, therebyinterfering with the accuracy of an assay for the drug. A primary factoraffecting bioavailability and of concern in the present methods is thepresence in a sample of drug metabolites that bind to antibody for thedrug and render an assay for the drug inaccurate, particularly wherethere is minimal or no separation of such metabolites from the drug andminimal or no separation of other components in a sample. The term“bioavailability” as used herein with respect to a “hydrophobicantibody” refers to the amount of hydrophobic antibody that is employedin an assay and that is available for binding to an analyte.

In accordance with the present embodiments, “enhanced bioavailability”or “enhancement of bioavailability” or “enhance the bioavailability”with respect to a hydrophobic drug means that there is an enhancement orincrease in the amount of the hydrophobic drug available for detectionin a sample that contains metabolites of the drug that cross-react withan antibody for the drug. In accordance with the present embodiments,“enhanced bioavailability” or “enhancement of bioavailability” or“enhance the bioavailability” with respect to a hydrophobic antibodymeans that there is an enhancement or increase in the amount of thehydrophobic antibody available for binding to an analyte.

In accordance with the present embodiments, “selectively enhancedbioavailability” or “selective enhancement of bioavailability” or“selectively enhance the bioavailability” with respect to a hydrophobicdrug means that there is an enhancement or increase in the amount of thehydrophobic drug available for binding to an antibody for thehydrophobic drug and, therefore, for detection in a sample, relative tothe amount of metabolites of the hydrophobic drug that cross-react withan antibody for the drug and that are available for cross-reacting withthe antibody for the hydrophobic drug.

The phrase “at least” as used herein means that the number of specifieditems may be equal to or greater than the number recited. The phrase“about” as used herein means that the number recited may differ by plusor minus 10%; for example, “about 5” means a range of 4.5 to 5.5.

Accordingly, as mentioned above, an embodiment of the present inventionis a method for selectively enhancing the bioavailability of ahydrophobic drug over metabolites of the hydrophobic drug. A combinationis formed in a medium where the combination comprises the sample, areleasing agent, and a selective solubility agent for the hydrophobicdrug. The releasing agent displaces the hydrophobic drug, and itsmetabolites, from endogenous binding moieties. The selective solubilityagent promotes equalization of the bioavailability of the hydrophobicdrug and that of the metabolites in the medium. The selective solubilityagent comprises a water miscible, non-volatile organic solvent and ispresent in the medium in a concentration sufficient to selectivelyenhance the bioavailability of the hydrophobic drug over that of themetabolites in the medium.

The sample to be analyzed is one that is suspected of containing one ormore hydrophobic drug analytes. The sample typically comprises one ormore endogenous binding moieties that bind to the hydrophobic drug. Theendogenous binding moieties may be binding proteins that bind ahydrophobic drug such as a lipoprotein, e.g., a protein that comprises alipid moiety or other substances that bind the hydrophobic drug such ascholesterol, triglyceride, and so forth. The samples are preferably fromhumans or animals and include biological fluids such as whole blood,serum, plasma, sputum, lymphatic fluid, semen, vaginal mucus, feces,urine, spinal fluid, saliva, stool, cerebral spinal fluid, tears, mucus,and the like; biological tissue such as hair, skin, sections or excisedtissues from organs or other body parts; and so forth. In manyinstances, the sample is whole blood, plasma or serum and, in aparticular embodiment the sample is whole blood. The sample is notpretreated to remove such endogenous binding moieties.

The sample can be prepared in any convenient medium that does notinterfere with an assay; an aqueous medium generally is employed. Thenature of the medium is discussed in more detail below. A releasingagent and a selective solubility agent for the hydrophobic drug inaccordance with the present methods are combined in the medium, whichmay also include a hemolytic agent.

Hemolytic Agent

A hemolytic agent is a compound or mixture of compounds that disrupt theintegrity of the membranes of red blood cells thereby releasingintracellular contents of the cells. Numerous hemolytic agents are knownin the art. Hemolytic agents include, for example, non-ionic detergents,anionic detergents, amphoteric detergents, low ionic strength aqueoussolutions (hypotonic solutions), bacterial agents, antibodies that causecomplement dependent lysis, and the like. Non-ionic detergents that maybe employed as the hemolytic agent include both synthetic detergents andnatural detergents. Examples of synthetic detergents include TRITON™X-100, TRITON™ N-101, TRITON™ X-114, TRITON™ X-405, TRITON™ SP-135,TWEEN® 20 (polyoxyethylene (20) sorbitan monolaurate), TWEEN® 80(polyoxyethylene (20) sorbitan monooleate), DOWFAX®, ZONYL®,pentaerythrityl palmitate, ADOGEN® 464, ALKANOL® 6112 surfactant, allylalcohol 1,2-butoxylate-block-ethoxylate HLB 6, BRIJ®, ethylenediaminetetrakis(ethoxylate-block-propoxylate) tetrol, IGEPAL®, MERPOL®,poly(ethylene glycol),2-[ethyl[(heptadecafluorooctyl)sulfonyl]amino]ethyl ether,polyethylene-block-poly(ethylene glycol), polyoxyethylene sorbitantetraoleate, polyoxyethylene sorbitol hexaoleate, TERGITOL® NP-9, GAFAC®(RHODAFAC®, an alkyl polyoxyethylene glycol phosphate ester such as, forexample, alpha-dodecyl-omega-hydroxypoly(oxy-1,2-ethanediyl) phosphate),and EP110® and the like. Naturally-occurring detergents that may beemployed as the hemolytic agent include, for example, saponins, sodiumor potassium neutralized fatty acid, neutralized phospholipids,diacylglycerol, neutralized phosphatidyl serine, phosphatidate,neutralized phosphatidyl ethanolamine, phosphatidyl choline,phosphatidyl inositol, phosphatidylcholine, bile salt, unesterifiedcholesterol, neutralized sphingosine, ceramide, and the like.Combinations of one or more synthetic detergents or one or morenaturally occurring detergents and combinations of synthetic detergentsand naturally occurring detergents may also be employed.

The nature and amount or concentration of hemolytic agent employeddepends on the nature of the sample, the nature of the hydrophobic drug,the nature of the rest of the reagent components, the reactionconditions, and the like. The amount of the hemolytic agent is at leastsufficient to cause lysis of red blood cells to release contents of thecells. In some embodiments the amount of the hemolytic agent is about0.0001% to about 0.5%, about 0.001% to about 0.4%, about 0.01% to about0.3%, about 0.01% to about 0.2%, about 0.1% to about 0.3%, about 0.2% toabout 0.5%, about 0.1% to about 0.2%, and so forth (percent isweight/volume).

Releasing Agent

The releasing agent displaces the hydrophobic drug from endogenousbinding moieties. The releasing agent can, and does in many instances,displace metabolites of the hydrophobic drug from endogenous bindingmoieties. In many embodiments the releasing agent has high bindingaffinity to the endogenous binding proteins so that it readily displacesthe hydrophobic drug, and its metabolites, from endogenous bindingproteins. In addition, the releasing agent does not bind to anysignificant degree to an antibody for the drug that is used in theassay. By the phrase “does not bind to any significant degree” is meantthat the extent of binding should be low enough so that an accurateassay for the drug may be carried out. The releasing agent may be anymoiety, either a single compound or a mixture of compounds, whichaccomplishes the desired result of displacement with no significantbinding to an assay antibody. In many embodiments the releasing agentdisplaces the hydrophobic drug and its metabolite from endogenousbinding substances to render both the hydrophobic drug and themetabolites substantially equally accessible to an antibody for thehydrophobic drug. “Substantially equally accessible” means that theamount of hydrophobic drug available for binding to antibody does notvary to any significant extent from the total amount of metabolites ofthe hydrophobic drug that are available for binding to the antibody. Theamount of metabolites available for binding to an antibody for thehydrophobic drug is dependent on considerations such as, for example,the binding affinity of particular metabolites for the antibody for thehydrophobic drug. The above percentages are based on the assumption thatthe drug metabolites have approximately the same binding affinity forthe antibody for the hydrophobic drug as the hydrophobic drug itself.Otherwise, the above percentages should be adjusted based on the actualbinding affinity of the hydrophobic drug metabolites.

In some embodiments the releasing agent is an analog, includingstructural analogs, of the hydrophobic drug. A hydrophobic drug analogis a modified drug that can displace the analogous hydrophobic drug froma binding protein but does not compete to any substantial degree for areceptor such as an antibody for the hydrophobic drug. The modificationprovides means to join a hydrophobic drug analog to another molecule.The hydrophobic drug analog will usually differ from the hydrophobicdrug by more than replacement of a hydrogen with a bond which links thedrug analog to a hub or label, but need not. The hydrophobic drug analogmay be, for example, the hydrophobic drug conjugated to another moleculethrough a linking group, and so forth. For hydrophobic drugs thatcomprise a hydroxy or carboxylic acid functionality, the releasing agentmay be an ester of the hydrophobic drug, which has a high bindingaffinity for endogenous binding proteins relative to the hydrophobicdrug to be detected and which has no significant binding affinity for anantibody for the hydrophobic drug. For example, in a determination forsirolimus, an ester of sirolimus may be employed as the releasing agentso long as it meets the above requirements. A structural analog is amoiety that has the same or similar structural or spatialcharacteristics as the hydrophobic drug such that the structural analogaccomplishes the same or similar result as the analog of the hydrophobicdrug. The structural analog may be, for example, another compound thatis related to the hydrophobic drug. For example, in a determination forsirolimus, an ester of tacrolimus may be employed as the releasingagent. The ester may be, for example, a carbamate, a carbonate, an esterof a C₁ to C₆ carboxylic acid, and the like. See, for example, U.S. Pat.No. 7,186,518, the relevant disclosure of which is incorporated hereinby reference. Other examples of releasing agents include [Thr₂, Leu₅,D-Hiv₈, Leu₁₀]-cyclosporin A for cyclosporin A, FK506 for sirolimus,sirolimus for FK506, and the like. See, for example, U.S. Pat. No.6,187,547, the relevant disclosure of which is incorporated herein byreference.

The concentration of the releasing agent in the medium is thatsufficient to achieve the desired result of displacing the hydrophobicdrug, and in many instances the metabolites of the hydrophobic drug,from endogenous binding moieties to render the drug and metabolitesaccessible for binding to an antibody for the drug as discussed above.The amount or concentration of the releasing agent employed depends onthe nature of the sample, the nature of the hydrophobic drug, the natureof the drug metabolites, the nature of other reagent components, thereaction conditions and the like. In some embodiments the amount of thereleasing agent is about 0.000001% to about 0.5%, about 0.0001% to about0.4%, about 0.001% to about 0.3%, about 0.01% to about 0.2%, about 0.1%to about 0.3%, about 0.2% to about 0.5%, about 0.1% to about 0.2%, andso forth (percent is weight/volume).

Selective Solubility Agent

The selective solubility agent promotes equalization of thebioavailability of the hydrophobic drug and that of the metabolites inthe medium. The nature of the selective solubility agent is such as toprovide a quasi-hydrophobic milieu to dissolve drug and/or drugmetabolites, which are released from endogenous binding moieties by thereleasing agent. By using the selective solubility agent, the drug andmetabolite are made similarly accessible to the assay antibody,resulting in reduction of metabolite cross-reactivity that would beotherwise higher due to its lower protein binding and higher solubilityin a pure aqueous solution compared to the parent drug. The presence ofa selective solubility agent in accordance with the embodiments hereinensures that both the released drug and metabolites are substantiallyequally dissolved in the pretreatment medium and/or in the assay medium.In some embodiments the selective solubility agent enhances thebioavailability of a hydrophobic antibody that is employed in an assay.

The selective solubility agent comprises a water miscible, non-volatileorganic solvent and is usually a liquid at room temperature (about 18°C. to about 23° C.). The selective solubility agent should comprise ahydrophilic or polar region in the molecule for it to be miscible withwater and a hydrophobic or non-polar region so that it can dissolvehydrophobic drugs and, therefore, has a hydrophobic drug-dissolvingcapability. Nevertheless, the overall polarity is such that the organicsolvent is miscible with water. The organic solvent is miscible withwater when it is capable of dissolving in water in all proportions at atemperature of about 1 to about 50° C. The organic solvents that may beused as the selective solubility agent have unlimited solubility in anaqueous medium.

As mentioned above, the selective solubility agent, when added to thepretreatment medium or assay medium, assists in dissolving hydrophobicdrugs in the medium. The extent to which a hydrophobic drug dissolves ina medium that contains the selective solubility agent versus the samemedium in the absence of the selective solubility agent is greater thanabout 80%, or greater than about 85%, or greater than about 90%, orgreater than about 95%, or about 100% or greater. For example, if 2ng/mL of hydrophobic drug dissolved in a medium without the selectivesolubility agent and 4 ng/mL of hydrophobic drug dissolved in the samemedium that contained the selective solubility in the appropriate range,the increase would be 100%.

The term “nonvolatile” means that the organic solvent has a vaporpressure as low as or lower than pure water at a given temperature, and,after combination with water in a percentage in accordance with thepresent methods, the resulting medium has a vapor pressure, at a giventemperature, as low as or lower than that of pure water. For example,the vapor pressure of DMSO at 8° C. is 21.7 Pa and the vapor pressure ofwater at 8° C. is 1044 Pa; the vapor pressure of 15% DMSO in water is978 Pa, which is lower than that of the pure water. On the other hand,ethanol, for example, would not be a suitable selective solubility agentin accordance with the present methods because its vapor pressure at 8°C. is 2754 Pa, much higher than that of pure water. The vapor pressureof a mixture of 10% ethanol in water has a vapor pressure of 1115 Pa,which is higher than that of pure water.

In addition to carbon and hydrogen, the organic solvent employed as theselective solubility agent may contain one or more of oxygen, sulfur,nitrogen, phosphorus, which may be present in various combinations toform functionalities such as, for example, hydroxyl, amine, amide,thiol, sulfoxide, sulfone, phosphate, phosphite, carboxylic acid ester,ether, and so forth. In some embodiments the selective solubility agentcontains 2 carbon atoms, or 3 carbon atoms, or 4 carbon atoms, or 5carbon atoms, or 6 carbon atoms as well as one or more of the abovefunctionalities. Embodiments of the selective solubility agent include,by way of illustration and not limitation, C₂, or C₃, or C₄, or C₅, orC₆ polyols comprising 2 hydroxy groups or 3 hydroxy groups such as, forexample, ethylene glycol, propylene glycol, glycerol, and the like, C₂,or C₃, or C₄, or C₅, or C₆ sulfoxides such as, for example, dimethylsulfoxide, diethyl sulfoxide, and so forth, C₂, or C₃, or C₄, or C₅, orC₆ sulfones such as, for example, dimethyl sulfone, diethyl sulfone, andso forth, C₂, or C₃, or C₄, or C₅, or C₆ amides such as, for example,formamides, e.g., dimethyl formamide, diethyl formamide,N-methylpyrrolidone, tetramethyl urea, dimethylacetamide, and so forth,C₂ to C₆ mono-, di- and tri-ethers of a polyol comprising 2 hydroxygroups or 3 hydroxy groups such as, for example, 1-methoxy-2-propanol,1,2-dimethoxy propanol, and so forth and C₂ to C₆ mono-, di- andtri-esters of a polyol comprising 2 hydroxy groups or 3 hydroxy groupssuch as, for example, 2-hydroxypropyl acetate, bis(2-methoxyethyl)ether(diglyme), and so forth. The selective solubility agent may be a singleorganic solvent or a combination of organic solvents having theaforementioned properties.

The concentration of the selective solubility agent in the medium issufficient to achieve selective enhancement of the bioavailability ofthe hydrophobic drug, over that of the metabolites, in the medium.Selective enhancement of the bioavailability of the hydrophobic drugover that of its metabolites is achieved when the amount of hydrophobicdrug that is detectable is increased over that obtained in the absenceof the selective solubility agent by at least about 50%, by at leastabout 75%, by at least about 90%, by at least about 100%, by at leastabout 125%, by at least about 150%, by at least about 175%, by at leastabout 200%, by at least about 225%, by at least about 250%, by at leastabout 275%, by at least about 300%, by at least about 325%, by at leastabout 350%, by at least about 375%, by at least about 400%, and soforth. In other words, selective enhancement of the bioavailability ofthe hydrophobic drug over that of its metabolites is achieved when theamount of hydrophobic drug that is detectable is increased over thatobtained in the absence of the selective solubility agent by about 0.5to about 4 times, or about 0.75 to about 4 times, or about 1 to about 4times, or about 0.5 to about 3.5 times, or about 0.5 to about 3 times,or about 0.5 to about 2.5 times, or about 0.5 to about 2 times, or about0.75 to about 3.5 times, or about 0.75 to about 3 times, or about 0.75to about 2.5 times, or about 0.75 to about 2 times, or about 1 to about3.5 times, or about 1 to about 3 times, or about 1 to about 2.5 times,or about 1 to about 2 times, and so forth.

The amount or concentration of selective solubility agent employeddepends on the nature of the sample, the nature of the hydrophobic drug,the nature of the organic solvent, the nature of other reagentcomponents, the reaction conditions, whether the medium is apretreatment medium or an assay medium, and the like. In someembodiments the amount of the selective solubility agent in apretreatment medium is about 10% to about 30%, about 11% to about 25%,about 12% to about 20%, about 13% to about 19%, about 14% to about 18%,about 15% to about 17%, about 15% to about 25%, about 16% to about 24%,about 17% to about 23%, about 18% to about 22%, about 19% to about 21%,about 15% to about 20%, about 16% to about 19%, and so forth (volume tovolume). In some embodiments the amount of the selective solubilityagent in an assay medium is about 1.0% to about 10%, about 2.0% to about9.0%, about 2.1% to about 8.0%, about 2.2% to about 7.0%, about 2.3% toabout 6.0%, about 2.4% to about 5%, about 2.5% to about 4.5%, about 3.0%to about 6.0%, about 3.1% to about 5.0%, about 3.2% to about 4.9%, about3.3% to about 4.8%, about 3.4% to about 4.7%, about 3.5% to about 4.5%,and so forth (volume to volume).

Pretreatment of Sample

The sample, a hemolytic agent (if employed), the releasing agent and theselective solubility agent are combined in a medium, which, as mentionedabove, is usually an aqueous medium and is referred to herein as apretreatment medium. All of the above may be combined simultaneously inthe medium or one or more of the above reagents may be addedsequentially in concentrations as discussed above. The medium may alsocomprise one or more preservatives as are known in the art such as, forexample, sodium azide, neomycin sulfate, PROCLIN® 300, Streptomycin, andthe like. The pH for the medium will usually be in the range of about 4to about 11, more usually in the range of about 5 to about 10, andpreferably in the range of about 6.5 to about 9.5.

Various buffers may be used to achieve the desired pH and maintain thepH during the incubation period. Illustrative buffers include borate,phosphate, carbonate, tris, barbital, PIPES, HEPES, MES, ACES, MOPS,BICINE, and the like. The medium may also comprise agents for preventingthe formation of blood clots. Such agents are well known in the art andinclude, for example, EDTA, EGTA, citrate, heparin, and the like.Various ancillary materials may be employed in the above methods. Forexample, in addition to buffers and preservatives, the medium maycomprise stabilizers for the medium and for the reagents employed. Allof the above materials are present in a concentration or amountsufficient to achieve the desired effect or function.

The medium is incubated under conditions for hemolyzing cells in thesample, for releasing the hydrophobic drug and its metabolites fromendogenous binding moieties and for enhancing the bioavailability of thehydrophobic drug. The incubation period may be about 1 second to about60 minutes, or about 1 second to about 6 minutes, or about 1 second toabout 5 minutes, or about 1 second to about 3 minutes, or about 1 secondto about 2 minutes, or about 1 second to about 1 minute, or about 1second to about 30 seconds, or about 1 second to about 20 seconds, orabout 1 second to about 10 seconds, or about 5 seconds to about 60minutes, or about 5 seconds to about 6 minutes, or about 5 seconds toabout 5 minutes, or about 5 seconds to about 3 minutes, or about 5seconds to about 2 minutes, or about 5 seconds to about 1 minute, orabout 5 seconds to about 30 seconds, or about 5 seconds to about 20seconds, or about 5 seconds to about 10 seconds, or about 10 seconds toabout 60 minutes, or about 10 seconds to about 6 minutes, or about 10seconds to about 5 minutes, or about 10 seconds to about 3 minutes, orabout 10 seconds to about 2 minutes, or about 10 seconds to about 1minute, or about 10 seconds to about 30 seconds, or about 10 seconds toabout 20 seconds, or about 20 seconds to about 60 minutes, or about 20seconds to about 6 minutes, or about 20 seconds to about 5 minutes, orabout 20 seconds to about 3 minutes, or about 20 seconds to about 2minutes, or about 20 seconds to about 1 minute, or about 20 seconds toabout 30 seconds, or about 30 seconds to about 60 minutes, or about 30seconds to about 6 minutes, or about 30 seconds to about 5 minutes, orabout 30 seconds to about 3 minutes, or about 30 seconds to about 2minutes, or about 30 seconds to about 1 minute, or about 1 minute toabout 30 minutes, or about 1 minute to about 20 minutes, or about 1minute to about 10 minutes, or the like.

The temperature during the incubation is usually about 10° C. to about45° C., or about 10° C. to about 35° C., or about 10° C. to about 25°C., or about 15° C. to about 45° C., or about 15° C. to about 35° C., orabout 15° C. to about 25° C., or about 20° C. to about 45° C., or about20° C. to about 35° C., or about 20° C. to about 25° C., or the like.

General Description of Assays for a Hydrophobic Drug

Following the above incubation period, reagents for determining thepresence and/or amount of the hydrophobic drug in the sample are addedto the medium. The nature of the reagents is dependent on the particulartype of assay to be performed. In general, the assay is a method for thedetermination or measuring of the presence and/or amount of ahydrophobic analyte. Various assay methods are discussed below by way ofillustration and not limitation.

In many embodiments the reagents comprise at least one antibody for thehydrophobic drug. By the phrase “antibody for the hydrophobic drug” ismeant an antibody that binds specifically to the hydrophobic drug anddoes not bind to any significant degree to other substances that woulddistort the analysis for the hydrophobic drug.

Antibodies specific for a hydrophobic drug for use in immunoassays canbe monoclonal or polyclonal. Such antibodies can be prepared bytechniques that are well known in the art such as immunization of a hostand collection of sera (polyclonal) or by preparing continuous hybridcell lines and collecting the secreted protein (monoclonal) or bycloning and expressing nucleotide sequences or mutagenized versionsthereof coding at least for the amino acid sequences required forspecific binding of natural antibodies.

Antibodies may include a complete immunoglobulin or fragment thereof,which immunoglobulins include the various classes and isotypes, such asIgA, IgD, IgE, IgG1, IgG2a, IgG2b and IgG3, IgM, etc. Fragments thereofmay include Fab, Fv and F(ab′)₂, Fab′, and the like. In addition,aggregates, polymers, and conjugates of immunoglobulins or theirfragments can be used where appropriate so long as binding affinity fora particular molecule is maintained.

Antiserum containing antibodies (polyclonal) is obtained bywell-established techniques involving immunization of an animal, such asa rabbit, guinea pig, or goat, with an appropriate immunogen andobtaining antisera from the blood of the immunized animal after anappropriate waiting period. State-of-the-art reviews are provided byParker, Radioimmunoassay of Biologically Active Compounds, Prentice-Hall(Englewood Cliffs, N.J., U.S., 1976), Butler, J. Immunol. Meth. 7: 1-24(1975); Broughton and Strong, Clin. Chem. 22: 726-732 (1976); andPlayfair, et al., Br. Med. Bull. 30: 24-31 (1974).

Antibodies can also be obtained by somatic cell hybridizationtechniques, such antibodies being commonly referred to as monoclonalantibodies. Monoclonal antibodies may be produced according to thestandard techniques of Köhler and Milstein, Nature 265:495-497, 1975.Reviews of monoclonal antibody techniques are found in LymphocyteHybridomas, ed. Melchers, et al. Springer-Verlag (New York 1978), Nature266: 495 (1977), Science 208: 692 (1980), and Methods of Enzymology 73(Part B): 3-46 (1981).

In another approach for the preparation of antibodies, the sequencecoding for antibody binding sites can be excised from the chromosome DNAand inserted into a cloning vector, which can be expressed in bacteriato produce recombinant proteins having the corresponding antibodybinding sites.

As discussed above, an antibody selected for use in an immunoassay for ahydrophobic drug, for example, should specifically and preferentiallybind the hydrophobic drug and its pharmaceutically active metabolitesover other ligands such as other metabolites or related drugs. Forexample, an antibody for tacrolimus should specifically andpreferentially bind tacrolimus over, e.g., rapamycin. In general, anantibody should be capable of distinguishing between one hydrophobicdrug relative to a second hydrophobic drug. At least about 5-fold, atleast about 10-fold, or at least about 20-fold, of the first hydrophobicdrug will be bound to the antibody if the antibody is combined with asample containing the hydrophobic drug. While the binding also dependson relative concentration of the hydrophobic drug, the binding will behigher for the first hydrophobic drug if the binding constant for thefirst hydrophobic drug is greater than the binding constant for thesecond hydrophobic drug, at least about 10-fold higher or at least about50-fold higher and up to 1000-fold or higher.

Other reagents are included in the assay medium depending on the natureof the assay to be conducted. Such assays usually involve reactionsbetween binding partners such as a hydrophobic drug analyte and acorresponding antibody or the binding between an antibody and acorresponding binding partner such as a second antibody that binds tothe first antibody. Accordingly, the binding partner may be a protein,which may be an antibody or an antigen. The binding partner may be amember of a specific binding pair (“sbp member”), which is one of twodifferent molecules, having an area on the surface or in a cavity, whichspecifically binds to and is thereby defined as complementary with aparticular spatial and polar organization of the other molecule. Themembers of the specific binding pair will usually be members of animmunological pair such as antigen-antibody, although other specificbinding pairs such as biotin-avidin, hormones-hormone receptors,enzyme-substrate, nucleic acid duplexes, IgG-protein A, polynucleotidepairs such as DNA-DNA, DNA-RNA, and the like are not immunological pairsbut are included within the scope of sbp member.

Accordingly, specific binding involves the specific recognition of oneof two different molecules for the other compared to substantially lessrecognition of other molecules. On the other hand, non-specific bindinginvolves non-covalent binding between molecules that is relativelyindependent of specific surface structures. Non-specific binding mayresult from several factors including hydrophobic interactions betweenmolecules. Preferred binding partners are antibodies.

Many types of immunoassays may be employed in the present methods todetermine the presence and/or amount of a hydrophobic drug analyte in asample suspected of containing such analytes. The immunoassays mayinvolve labeled or non-labeled reagents. Immunoassays involvingnon-labeled reagents usually comprise the formation of relatively largecomplexes involving one or more antibodies. Such assays include, forexample, immunoprecipitin and agglutination methods and correspondinglight scattering techniques such as, e.g., nephelometry andturbidimetry, for the detection of antibody complexes. Labeledimmunoassays include enzyme immunoassays, fluorescence polarizationimmunoassays, radioimmunoassay, inhibition assay, induced luminescence,fluorescent oxygen channeling assay, and so forth.

In many of the assays discussed herein, a label is employed; the labelis usually part of a signal producing system (“sps”). The nature of thelabel is dependent on the particular assay format. An sps usuallyincludes one or more components, at least one component being adetectable label, which generates a detectable signal that relates tothe amount of bound and/or unbound label, i.e. the amount of label boundor not bound to the hydrophobic drug being detected or to an agent thatreflects the amount of the hydrophobic drug to be detected. The label isany molecule that produces or can be induced to produce a signal, andmay be, for example, a fluorescer, radiolabel, enzyme, chemiluminesceror photosensitizer. Thus, the signal is detected and/or measured bydetecting enzyme activity, luminescence, light absorbance orradioactivity, and so forth, as the case may be.

Suitable labels include, by way of illustration and not limitation,enzymes such as alkaline phosphatase, glucose-6-phosphate dehydrogenase(“G6PDH”) and horseradish peroxidase; ribozyme; a substrate for areplicase such as QB replicase; promoters; dyes; fluorescers, such asfluorescein, isothiocyanate, rhodamine compounds, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine;complexes such as those prepared from CdSe and ZnS present insemiconductor nanocrystals known as Quantum dots; chemiluminescers suchas isoluminol; sensitizers; coenzymes; enzyme substrates; radiolabelssuch as ¹²⁵l, ¹³¹l, ¹⁴C, ³H, ⁵⁷Co and ⁷⁵Se; particles such as latexparticles, carbon particles, metal particles including magneticparticles, e.g., chromium dioxide (CrO₂) particles, and the like; metalsol; crystallite; liposomes; cells, etc., which may be further labeledwith a dye, catalyst or other detectable group. Suitable enzymes andcoenzymes are disclosed in Litman, et al., U.S. Pat. No. 4,275,149,columns 19-28, and Boguslaski, et al., U.S. Pat. No. 4,318,980, columns10-14; suitable fluorescers and chemiluminescers are disclosed inLitman, et al., U.S. Pat. No. 4,275,149, at columns 30 and 31; which areincorporated herein by reference.

The label can directly produce a signal and, therefore, additionalcomponents are not required to produce a signal. Numerous organicmolecules, for example fluorescers, are able to absorb ultraviolet andvisible light, where the light absorption transfers energy to thesemolecules and elevates them to an excited energy state. This absorbedenergy is then dissipated by emission of light at a second wavelength.Other labels that directly produce a signal include radioactive isotopesand dyes.

Alternately, the label may need other components to produce a signal,and the signal producing system would then include all the componentsrequired to produce a measurable signal. Such other components mayinclude substrates, coenzymes, enhancers, additional enzymes, substancesthat react with enzymic products, catalysts, activators, cofactors,inhibitors, scavengers, metal ions, and a specific binding substancerequired for binding of signal generating substances. A detaileddiscussion of suitable signal producing systems can be found in Ullman,et al., U.S. Pat. No. 5,185,243, columns 11-13, incorporated herein byreference.

The label or other sps members can be bound to a support. A hydrophobicdrug derivative or analog may be bound to a solid support in any mannerknown in the art, provided only that the binding does not substantiallyinterfere with the analogs ability to bind with an antibody. In someembodiments, the hydrophobic drug derivative or analog may be coated orcovalently bound directly to the solid phase or may have layers of oneor more carrier molecules such as poly(amino acids) including proteinssuch as serum albumins or immunoglobulins, or polysaccharides(carbohydrates) such as, for example, dextran or dextran derivatives.Linking groups may also be used to covalently couple the solid supportand the hydrophobic drug. Other methods of binding the hydrophobic drugderivatives are also possible. For instance, a solid support may have acoating of a binder for a small molecule such as, for example, avidin,an antibody, etc., and a small molecule such as, e.g., biotin, hapten,etc., can be bound to the hydrophobic drug derivative or vice versa. Thebinding of components to the surface of a support may be direct orindirect, covalent or non-covalent and can be accomplished by well-knowntechniques, commonly available in the literature. See, for example,“Immobilized Enzymes,” Ichiro Chibata, Halsted Press, New York (1978)and Cautrecasas, J. Biol. Chem., 245:3059 (1970).

The support may be comprised of an organic or inorganic, solid or fluid,water insoluble material, which may be transparent or partiallytransparent. The support can have any of a number of shapes, such asparticle, including bead, film, membrane, tube, well, strip, rod, planarsurfaces such as, e.g., plate, paper, etc., fiber, and the like.Depending on the type of assay, the support may or may not besuspendable in the medium in which it is employed. Examples ofsuspendable supports are polymeric materials such as latex, lipidbilayers or liposomes, oil droplets, cells and hydrogels, magneticparticles, and the like. Other support compositions include polymers,such as nitrocellulose, cellulose acetate, poly(vinyl chloride),polyacrylamide, polyacrylate, polyethylene, polypropylene,poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethyleneterephthalate), nylon, poly(vinyl butyrate), etc.; either used bythemselves or in conjunction with other materials.

The support may be a particle. The particles should have an averagediameter of at least about 0.02 microns and not more than about 100microns. In some embodiments, the particles have an average diameterfrom about 0.05 microns to about 20 microns, or from about 0.3 micronsto about 10 microns. The particle may be organic or inorganic, swellableor non-swellable, porous or non-porous, preferably of a densityapproximating water, generally from about 0.7 g/mL to about 1.5 g/mL,and composed of material that can be transparent, partially transparent,or opaque. The particles can be biological materials such as cells andmicroorganisms, e.g., erythrocytes, leukocytes, lymphocytes, hybridomas,streptococcus, Staphylococcus aureus, E. coli, viruses, and the like.The particles can also be particles comprised of organic and inorganicpolymers, liposomes, latex particles, magnetic or non-magneticparticles, phospholipid vesicles, chylomicrons, lipoproteins, and thelike. In some embodiments, the particles are chromium dioxide (chrome)particles or latex particles.

The polymer particles can be formed of addition or condensationpolymers. The particles will be readily dispersible in an aqueous mediumand can be adsorptive or functionalizable so as to permit conjugation toa hydrophobic drug analog, either directly or indirectly through alinking group. The particles can also be derived from naturallyoccurring materials, naturally occurring materials that aresynthetically modified, and synthetic materials. Among organic polymersof particular interest are polysaccharides, particularly cross-linkedpolysaccharides, such a agarose, which is available as Sepharose,dextran, available as Sephadex and Sephacryl, cellulose, starch, and thelike; addition polymers, such as polystyrene, polyvinyl alcohol,homopolymers and copolymers of derivatives of acrylate and methacrylate,particularly esters and amides having free hydroxyl functionalities, andthe like.

The label and/or other sps member may be bound to an sbp member oranother molecule. For example, the label can be bound covalently to ansbp member such as, for example, an antibody; a receptor for anantibody, a receptor that is capable of binding to a small moleculeconjugated to an antibody, or a ligand analog. Bonding of the label tothe sbp member may be accomplished by chemical reactions that result inreplacing a hydrogen atom of the label with a bond to the sbp member ormay include a linking group between the label and the sbp member. Othersps members may also be bound covalently to sbp members. For example,two sps members such as a fluorescer and quencher can each be bound to adifferent antibody that forms a specific complex with the analyte.Formation of the complex brings the fluorescer and quencher in closeproximity, thus permitting the quencher to interact with the fluorescerto produce a signal. Methods of conjugation are well known in the art.See, for example, Rubenstein, et al., U.S. Pat. No. 3,817,837,incorporated herein by reference.

Enzymes of particular interest as label proteins are redox enzymes,particularly dehydrogenases such as glucose-6-phosphate dehydrogenase,lactate dehydrogenase, etc., and enzymes that involve the production ofhydrogen peroxide and the use of the hydrogen peroxide to oxidize a dyeprecursor to a dye. Particular combinations include saccharide oxidases,e.g., glucose and galactose oxidase, or heterocyclic oxidases, such asuricase and xanthine oxidase, coupled with an enzyme which employs thehydrogen peroxide to oxidize a dye precursor, that is, a peroxidase suchas horse radish peroxidase, lactoperoxidase, or microperoxidase.Additional enzyme combinations are known in the art. When a singleenzyme is used as a label, other enzymes may find use such ashydrolases, transferases, and oxidoreductases, preferably hydrolasessuch as alkaline phosphatase and beta-galactosidase. Alternatively,luciferases may be used such as firefly luciferase and bacterialluciferase.

Illustrative co-enzymes that find use include NAD[H], NADP[H], pyridoxalphosphate, FAD[H], FMN[H], etc., usually coenzymes involving cyclingreactions. See, for example, U.S. Pat. No. 4,318,980, the disclosure ofwhich is incorporated herein by reference.

With label proteins such as, for example, enzymes, the molecular weightrange will be from about 10,000 to about 600,000, or from about 10,000to about 300,000 molecular weight. There is usually at least about 1hydrophobic drug analog per about 200,000 molecular weight, or at leastabout 1 per about 150,000 molecular weight, or at least about 1 perabout 100,000 molecular weight, or at least about 1 per about 50,000molecular weight, and so forth. In the case of enzymes, the number ofhydrophobic drug analog groups is usually from 1 to about 20, about 2 toabout 15, about 3 to about 12, or about 6 to about 10.

The term “non-poly(amino acid) labels” includes those labels that arenot proteins (e.g., enzymes). The non-poly(amino acid) label is capableof being detected directly or is detectable through a specific bindingreaction that produces a detectable signal. The non-poly(amino acid)labels include, for example, radioisotopes, luminescent compounds,supports, e.g., particles, plates, beads, etc., polynucleotides, and thelike. More particularly, the non-poly(amino acid) label can be isotopicor non-isotopic, usually non-isotopic, and can be a polynucleotidecoding for a catalyst, promoter, dye, coenzyme, enzyme substrate,radioactive group, a small organic molecule (including, e.g., biotin,fluorescent molecules, chemiluminescent molecules, and the like),amplifiable polynucleotide sequence, a support such as, for example, aparticle such as latex or carbon particle or chromium dioxide (chrome)particle or the like, metal sol, crystallite, liposome, cell, etc.,which may or may not be further labeled with a dye, catalyst or otherdetectable group, and the like.

One general group of immunoassays that may be employed includesimmunoassays using a limited concentration of antibody. Another group ofimmunoassays involves the use of an excess of one or more of theprincipal reagents such as, for example, an excess of an antibody forthe immunosuppressant drug. Another group of immunoassays areseparation-free homogeneous assays in which the labeled reagentsmodulate the label signal upon hydrophobic drug-antibody bindingreactions. Another group of assays includes labeled antibody reagentlimited competitive assays for hydrophobic drug that avoid the use ofproblematic labeled haptens. In this type of assay, the solid phaseimmobilized hydrophobic drug analyte is present in a constant, limitedamount. The partition of a label between the immobilized hydrophobicdrug analyte and free hydrophobic drug analyte depends on theconcentration of analyte in the sample.

The assays can be performed either without separation (homogeneous) orwith separation (heterogeneous) of any of the assay components orproducts. Homogeneous immunoassays are exemplified by the EMIT® assay(Syva Company, San Jose, Calif.) disclosed in Rubenstein, et al., U.S.Pat. No. 3,817,837, column 3, line 6 to column 6, line 64;immunofluorescence methods such as those disclosed in Ullman, et al.,U.S. Pat. No. 3,996,345, column 17, line 59, to column 23, line 25;enzyme channeling immunoassays (“ECIA”) such as those disclosed inMaggio, et al., U.S. Pat. No. 4,233,402, column 6, line 25 to column 9,line 63; the fluorescence polarization immunoassay (“FPIA”) asdisclosed, for example, in, among others, U.S. Pat. No. 5,354,693; andso forth.

Other enzyme immunoassays are the enzyme modulate mediated immunoassay(“EMMIA”) discussed by Ngo and Lenhoff, FEBS Lett. (1980) 116:285-288;the substrate labeled fluorescence immunoassay (“SLFIA”) disclosed byOellerich, J. Clin. Chem. Clin. Biochem. (1984) 22:895-904; the combinedenzyme donor immunoassays (“CEDIA”) disclosed by Khanna, et al., Clin.Chem. Acta (1989) 185:231-240; homogeneous particle labeled immunoassayssuch as particle enhanced turbidimetric inhibition immunoassays(“PETINIA”), particle enhanced turbidimetric immunoassay (“PETIA”),etc.; and the like.

Other assays include the sol particle immunoassay (“SPIA”), the dispersedye immunoassay (“DIA”); the metalloimmunoassay (“MIA”); the enzymemembrane immunoassays (“EMIA”); luminoimmunoassays (“LIA”); and soforth. Other types of assays include immunosensor assays involving themonitoring of the changes in the optical, acoustic and electricalproperties of an antibody-immobilized surface upon the binding of ahydrophobic drug. Such assays include, for example, optical immunosensorassays, acoustic immunosensor assays, semiconductor immunosensor assays,electrochemical transducer immunosensor assays, potentiometricimmunosensor assays, amperometric electrode assays, and the like.

In one embodiment the assay is an induced luminescence immunoassay,which is described in U.S. Pat. No. 5,340,716 (Ullman, et al.) entitled“Assay Method Utilizing Photoactivated Chemiluminescent Label” (“inducedluminescence assay”), which disclosure is incorporated herein byreference. In one approach the assay uses a particle incorporating aphotosensitizer and a label particle incorporating a chemiluminescentcompound. The label particle is conjugated to an sbp member, forexample, an antibody for the hydrophobic drug that is capable of bindingto the hydrophobic drug analyte to form a complex, or to a second sbpmember to form a complex, in relation to the presence of the hydrophobicdrug analyte. If the hydrophobic drug analyte is present, thephotosensitizer and the chemiluminescent compound come into closeproximity. The photosensitizer generates singlet oxygen and activatesthe chemiluminescent compound when the two labels are in closeproximity. The activated chemiluminescent compound subsequently produceslight. The amount of light produced is related to the amount of thecomplex formed, which in turn is related to the amount of hydrophobicdrug analyte present.

By way of further illustration, chemiluminescent particles are employed,which comprise the chemiluminescent compound associated therewith suchas by incorporation therein or attachment thereto. An sbp member thatbinds to the hydrophobic drug analyte, such as, for example, an antibodyfor a hydrophobic drug, is bound to a polysaccharide coating theparticles. A second sbp member that binds to the hydrophobic druganalyte is part of a biotin conjugate. Streptavidin is conjugated to asecond set of particles having a photosensitizer associated therewith.The binding of the streptavidin to this second set of particles(photosensitizer particles) may or may not involve a polysaccharide onthe particles. The chemiluminescent particles are mixed with a samplesuspected of containing a hydrophobic drug analyte and thephotosensitizer particles. The reaction medium is incubated to allow theparticles to bind to the hydrophobic drug analyte by virtue of thebinding of the sbp members to the hydrophobic drug analyte. Then, themedium is irradiated with light to excite the photosensitizer, which iscapable in its excited state of activating oxygen to a singlet state.Because the chemiluminescent compound of one of the sets of particles isnow in close proximity to the photosensitizer by virtue of the presenceof the hydrophobic drug analyte, it is activated by singlet oxygen andemits luminescence. The medium is then examined for the presence and/orthe amount of luminescence or light emitted, the presence thereof beingrelated to the presence and/or amount of the hydrophobic drug analyte.

Another particular example of an assay that may be employed for thedetermination of a hydrophobic drug analyte is discussed in U.S. Pat.No. 5,616,719 (Davalian, et al.), which describes fluorescent oxygenchanneling immunoassays.

In some embodiments multi-analyte immunoassays may be utilized where thehydrophobic drug analyte may be the subject of detection along with oneor more other analytes such as other drugs and the like. Suchmulti-analyte systems are described, for example, in Loor, et al., J.Anal. Toxicol. 12: 299 (1988).

The assays discussed above are normally carried out in an aqueousbuffered medium at a moderate pH, generally that which provides optimumassay sensitivity. The pH for the assay medium will usually be in therange of about 4 to about 11, more usually in the range of about 5 toabout 10, and preferably in the range of about 6.5 to about 9.5. The pHwill usually be a compromise between optimum binding of the bindingmembers of any specific binding pairs, the pH optimum for other reagentsof the assay such as members of the signal producing system, and soforth.

Various buffers may be used to achieve the desired pH and maintain thepH during the determination. Illustrative buffers include borate,phosphate, carbonate, tris, barbital and the like. The particular bufferemployed is not critical, but in an individual assay one or anotherbuffer may be preferred. Various ancillary materials may be employed inthe above methods. For example, in addition to buffers the medium maycomprise stabilizers for the medium and for the reagents employed.Frequently, in addition to these additives, proteins may be included,such as albumins; quaternary ammonium salts; polyanions such as dextransulfate; binding enhancers, or the like.

One or more incubation periods may be applied to the medium at one ormore intervals including any intervals between additions of variousreagents mentioned above. The medium is usually incubated at atemperature and for a time sufficient for binding of various componentsof the reagents to occur. Moderate temperatures are normally employedfor carrying out the method and usually constant temperature,preferably, room temperature, during the period of the measurement.Incubation temperatures normally range from about 5° to about 99° C.,usually from about 15° C. to about 70° C., more usually 20° C. to about45° C. The time period for the incubation is about 0.2 seconds to about24 hours, or about 1 second to about 6 hours, or about 2 seconds toabout 1 hour, or about 1 to about 15 minutes. The time period depends onthe temperature of the medium and the rate of binding of the variousreagents, which is determined by the association rate constant, theconcentration, the binding constant and dissociation rate constant.Temperatures during measurements will generally range from about 10 toabout 50° C., or from about 15 to about 40° C.

The concentration of analyte that may be assayed generally varies fromabout 10⁻⁵ to about 10⁻¹⁷ M, more usually from about 10⁻⁶ to about 10⁻¹⁴M. Considerations, such as whether the assay is qualitative,semi-quantitative or quantitative (relative to the amount of hydrophobicdrug analyte present in the sample), the particular detection techniqueand the concentration of the analyte normally determine theconcentrations of the various reagents.

The concentrations of the various reagents in the assay medium willgenerally be determined by the concentration range of interest of thehydrophobic drug analyte, the nature of the assay, and the like.However, the final concentration of each of the reagents is normallydetermined empirically to optimize the sensitivity of the assay over therange. That is, a variation in concentration of hydrophobic drug analytethat is of significance should provide an accurately measurable signaldifference. Considerations such as the nature of the signal producingsystem and the nature of the analytes normally determine theconcentrations of the various reagents.

While the order of addition may be varied widely, there will be certainpreferences depending on the nature of the assay. The simplest order ofaddition is to add all the materials simultaneously and determine theeffect that the assay medium has on the signal as in a homogeneousassay. Alternatively, the reagents can be combined sequentially.Optionally, an incubation step may be involved subsequent to eachaddition as discussed above.

Examination Step

In a next step of the method in accordance with the present disclosure,the medium is examined for the presence of a complex comprising thehydrophobic drug and the antibody for the hydrophobic drug. The presenceand/or amount of the complex indicates the presence and/or amount of thehydrophobic drug in the sample.

The phrase “measuring the amount of a hydrophobic drug analyte” refersto the quantitative, semiquantitative and qualitative determination ofthe hydrophobic drug analyte. Methods that are quantitative,semiquantitative and qualitative, as well as all other methods fordetermining the hydrophobic drug analyte, are considered to be methodsof measuring the amount of the hydrophobic drug analyte. For example, amethod, which merely detects the presence or absence of the hydrophobicdrug analyte in a sample suspected of containing the hydrophobic druganalyte, is considered to be included within the scope of the presentinvention. The terms “detecting” and “determining,” as well as othercommon synonyms for measuring, are contemplated within the scope of thepresent invention.

In many embodiments the examination of the medium involves detection ofa signal from the medium. The presence and/or amount of the signal isrelated to the presence and/or amount of the hydrophobic drug in thesample. The particular mode of detection depends on the nature of thesps. As discussed above, there are numerous methods by which a label ofan sps can produce a signal detectable by external means, desirably byvisual examination, and include, for example, electromagnetic radiation,electrochemistry, heat, radioactivity detection, chemical reagents andso forth.

Activation of a signal producing system depends on the nature of thesignal producing system members. For those members of a signal producingsystem that are activated with light, the member is irradiated withlight. For members of signal producing systems that are on the surfaceof a particle, addition of a base may result in activation. Otheractivation methods will be suggested to those skilled in the art in viewof the disclosures herein. For some signal producing systems, no agentfor activation is necessary such as those systems that involve a labelthat is a radioactive label, an enzyme, and so forth. For enzymesystems, addition of a substrate and/or a cofactor may be necessary.

The examination for presence and/or amount of the signal also includesthe detection of the signal, which is generally merely a step in whichthe signal is read. The signal is normally read using an instrument, thenature of which depends on the nature of the signal. The instrument maybe a spectrophotometer, fluorometer, absorption spectrometer,luminometer, chemiluminometer, actinometer, photographic instrument, andthe like. The presence and amount of signal detected is related to thepresence and amount of the hydrophobic drug compound present in asample. Temperatures during measurements generally range from about 10°to about 70° C., or from about 20° to about 45° C., or about 20° toabout 25° C. In one approach standard curves are formed using knownconcentrations of the analytes to be screened. As discussed above,calibrators and other controls may also be used.

Specific Embodiments of Assays

The following examples describe specific embodiments of the invention byway of illustration and not limitation and are intended merely todescribe, and not to limit, the scope of the invention.

In a homogeneous assay after all of the reagents have been combined, thesignal is determined and related to the amount of analyte in the sample.For example, in an EMIT® assay for a hydrophobic drug, a samplesuspected of containing the hydrophobic drug is combined in an aqueousmedium either simultaneously or sequentially with an enzyme conjugate ofthe hydrophobic drug, i.e., an analog for the hydrophobic drug, andantibody capable of recognizing the hydrophobic drug. Generally, asubstrate for the enzyme is added, which results in the formation of achromogenic or fluorogenic product upon enzyme catalyzed reaction.Preferred enzymes are glucose-6-phosphate dehydrogenase and alkalinephosphatase but other enzymes may be employed. The hydrophobic druganalyte and the moieties of the enzyme conjugate compete for bindingsites on the antibody. The enzyme activity in the medium is thendetermined, usually by spectrophotometric means, and is compared to theenzyme activity determined when calibrators or reference samples aretested in which a known amount of the hydrophobic drug is present.Typically, the calibrators are tested in a manner similar to the testingof the sample suspected of containing the hydrophobic drug analytes. Thecalibrators typically contain differing, but known, concentrations ofthe hydrophobic drug analyte to be determined. Preferably, theconcentration ranges present in the calibrators span the range ofsuspected hydrophobic drug analyte concentrations in unknown samples.

The aforementioned assays may be carried out using mutantglucose-6-phosphate dehydrogenase as the enzyme of the enzyme conjugate.This mutant enzyme is described in U.S. Pat. Nos. 6,090,567 and6,033,890, the relevant disclosures of which are incorporated herein byreference. Furthermore, the assay may be conducted using antibodies forthe hydrophobic drug and using procedures as disclosed in U.S. Pat. Nos.5,328,828 and 5,135,863, the relevant disclosures of which areincorporated herein by reference.

Heterogeneous assays usually involve one or more separation steps andcan be competitive or non-competitive. A variety of competitive andnon-competitive assay formats are disclosed in Davalian, et al., U.S.Pat. No. 5,089,390, column 14, line 25 to column 15, line 9, whichdisclosure is incorporated herein by reference. In one type ofcompetitive assay, a support, as discussed herein, having antibodies forthe hydrophobic drug bound thereto is contacted with a medium containingthe sample and appropriate enzyme conjugates of the hydrophobic drug.After separating the support and the medium, the enzyme activity of thesupport or the medium is determined by conventional techniques andrelated to the presence and/or amount of the hydrophobic drug in thesample.

In certain embodiments a second enzyme may be employed in addition tothe enzyme of the enzyme conjugate. The enzymes of the pair of enzymesare related in that a product of the first enzyme serves as a substratefor the second enzyme.

Another embodiment of an assay format is a capture assay. In this assayformat, the antibody for the hydrophobic drug is covalently bound to amagnetic particle. The sample is incubated with these particles to allowthe hydrophobic drug in the sample to bind to the antibodies for thehydrophobic drug. Subsequently, an enzyme that has the hydrophobic drugor a derivative of the hydrophobic drug covalently attached is incubatedwith the magnetic particles. After washing, the amount of enzyme that isbound to the magnetic particles is measured and is inversely related tothe presence and/or amount of the hydrophobic drug in the sample.

The following specific assay descriptions are by way of illustration of,and not as a limitation on, the scope of the present invention.Selection of sirolimus as the hydrophobic drug is also by way ofillustration and not limitation as the present invention has generalapplication to detection of hydrophobic drugs in general andimmunosuppressant drugs in particular.

In one embodiment, the test sample or a sirolimus standard is mixed witha sirolimus conjugate, i.e., for example, an analog of sirolimus that isattached to biotin. The sirolimus of the test sample and the analog ofsirolimus are allowed to compete for binding to the antibody for thesirolimus, which is capable of binding to sirolimus or the analog ofsirolimus. After rinsing with an appropriate wash buffer, a detectionmolecule consisting of streptavidin or avidin conjugated to an enzyme,florescent or chemiluminescent molecule or radioactive moiety can beadded to the medium, which is then examined for the presence and/oramount of signal. The presence and/or amount of signal is related to thepresence and/or amount of sirolimus.

In one embodiment the assay employed is an induced luminescence assay asdescribed above. The reagents include two latex bead reagents and abiotinylated anti-sirolimus mouse monoclonal antibody. The first beadreagent is coated with sirolimus or a sirolimus analog and containschemiluminescent dye. The second bead reagent is coated withstreptavidin and contains a photosensitizer dye. In a first step, samplesuspected of containing sirolimus is incubated with biotinylatedantibody for sirolimus, which allows sirolimus from the sample tosaturate a fraction of the biotinylated antibody that is directlyrelated to the sirolimus concentration. In a second step, the first beadreagent is added and leads to the formation of bead/biotinylatedantibody immunocomplexes with the non-saturated fraction of thebiotinylated antibody. The second bead reagent is then added and bindsto the biotin to form bead pair immunocomplexes. When illuminated bylight at 680 nm, the second bead reagent converts dissolved oxygen inthe reaction solution into the more energetic singlet oxygen form. Inthe bead pairs, the singlet oxygen diffuses into the first bead reagentthereby triggering a chemiluminescent reaction. The resultingchemiluminescent signal is measured at 612 nm and is an inverse functionof the concentration of sirolimus in the sample. The amount of thissignal is related to the presence and or amount of sirolimus in thesample.

A specific example of another assay format is ACMIA (Affinity Chromiumdioxide Mediated Immuno Assay). For the ACMIA assay format, chromeparticles, which are coated with sirolimus or a sirolimus analog, areemployed as a first component. A second component is an antibody forsirolimus. This antibody, crosslinked to a reporter enzyme (for example,beta-galactosidase), is added to a reaction vessel in an excess amount,i.e., an amount greater than that required to bind all of the analytethat might be present in a sample. The antibody-enzyme conjugate ismixed with a sample suspected of containing sirolimus to allow thesirolimus analyte to bind to the antibody. Next, the chrome particlereagent is added to bind up any excess antibody-enzyme conjugate. Then,a magnet is applied, which pulls all of the chrome particles and excessantibody-enzyme out of the suspension, and the supernatant istransferred to a final reaction container. The substrate of the reporterenzyme is added to the final reaction container, and the enzyme activityis measured spectrophotometrically as a change in absorbance over time.The amount of this signal is related to the presence and/or amount ofsirolimus in the sample.

In a sandwich assay format, a first reagent comprising chrome particlescoated with anti-sirolimus antibodies and a second reagent comprising asecond antibody (or binding protein) for the first antibody conjugatedto a reporter enzyme are employed. In this format, the sample suspectedof containing sirolimus is incubated with the chrome particles so thatall of the sirolimus, if present in the sample, becomes bound to thechrome particles. The chrome particles are washed, using a magnet toseparate the bound analyte from the supernatant. Then, the secondreagent, i.e., antibody (or binding protein) conjugated to a reporterenzyme, is incubated with the chrome particles to form a “sandwich”.After washing, the amount of enzyme that is bound to the chrome ismeasured and is related to the presence and/or amount of sirolimus inthe sample.

Another assay format is EMIT® (Enzyme-Mediated Immunoassay Technology).In this assay format, an enzyme conjugate is formed such as, forexample, a conjugate of G-6-PDH and a sirolimus analog. An antibody forsirolimus is incubated with the enzyme-conjugate and a sample suspectedof containing sirolimus. Antibody for sirolimus binds to the sirolimusanalyte in the sample instead of binding to the enzyme conjugate, whichreduces the amount of inhibition of the enzyme activity that mightotherwise occur in the absence of sirolimus in the sample. In this way,samples with more sirolimus analyte will yield higher enzyme activity,and samples with no sirolimus analyte will have the maximum inhibitionand the lowest enzyme activity. The amount of reduction of inhibition ofenzyme activity is related to the amount of sirolimus in the sample.

The reagents for conducting a particular assay may be present in a kituseful for conveniently performing an assay for the determination of ahydrophobic drug analyte. In one embodiment a kit comprises in packagedcombination an antibody for a hydrophobic drug analyte and otherreagents for performing an assay, the nature of which depend upon theparticular assay format. The reagents may each be in separate containersor various reagents can be combined in one or more containers dependingon the cross-reactivity and stability of the reagents. The kit canfurther include other separately packaged reagents for conducting anassay such as additional sbp members, ancillary reagents such as anancillary enzyme substrate, and so forth.

The relative amounts of the various reagents in the kits can be variedwidely to provide for concentrations of the reagents that substantiallyoptimize the reactions that need to occur during the present method andfurther to optimize substantially the sensitivity of the assay. Underappropriate circumstances one or more of the reagents in the kit can beprovided as a dry powder, usually lyophilized, including excipients,which on dissolution will provide for a reagent solution having theappropriate concentrations for performing a method or assay inaccordance with the present invention. The kit can further include awritten description of a method in accordance with the present inventionas described above.

Other Embodiments

One embodiment of the present invention is a method for pretreating asample suspected of containing a hydrophobic drug for conducting anassay method for detecting the hydrophobic drug. A combination isprovided in a medium. The combination comprises (i) the sample, (ii) areleasing agent for releasing the hydrophobic drug and its metabolitesfrom endogenous binding moieties, and (iii) a selective solubility agentthat provides for substantially equal solubility of the hydrophobic drugand its metabolites in the medium. The selective solubility agentcomprises a water miscible, non-volatile organic solvent and is presentin the medium in a concentration sufficient to provide for substantiallyequal solubility of the hydrophobic drug and its metabolites in themedium. The medium is incubated under conditions for releasing thehydrophobic drug and its metabolites from endogenous binding moieties.

Another embodiment of the present invention is a method for determininga hydrophobic drug in a sample suspected of containing a hydrophobicdrug. A combination is provided in a medium. The combination comprises(i) the sample, (ii) a releasing agent for releasing the hydrophobicdrug and its metabolites from endogenous binding moieties, and (iii) aselective solubility agent that provides for substantially equalsolubility of the hydrophobic drug and its metabolites in the medium,wherein the selective solubility agent comprises a water miscible,non-volatile organic solvent and wherein the concentration of theselective solubility agent in the medium is sufficient to provide forsubstantially equal solubility of the hydrophobic drug and itsmetabolites in the medium. The combination in the medium furthercomprises a hemolytic agent. The medium is incubated under conditionsfor hemolyzing cells in the sample and for releasing the hydrophobicdrug and its metabolites from endogenous binding moieties. To the mediumis added reagents for determining the presence and/or amount of thehydrophobic drug in the sample wherein the reagents comprise at leastone antibody for the hydrophobic drug. The medium is examined for thepresence of a complex comprising the hydrophobic drug and the antibodyfor the hydrophobic drug, the presence and/or amount of the complexindicating the presence and/or amount of the hydrophobic drug in thesample.

Another embodiment of the present invention is a method for determiningan immunosuppressant drug in a sample suspected of containing animmunosuppressant drug. A combination is formed in a medium wherein thecombination comprises the sample, a releasing agent for releasing theimmunosuppressant drug and its metabolites from endogenous bindingmoieties and a selective solubility agent for the immunosuppressant drugand its metabolites. The selective solubility agent comprises a watermiscible, non-volatile organic solvent. The concentration of theselective solubility agent in the medium is sufficient to provide forsubstantially equal solubility of the immunosuppressant drug and itsmetabolites in the medium. The medium is incubated under conditions forreleasing the immunosuppressant drug and its metabolites from endogenousbinding moieties. To the medium is added (i) a reagent comprising (I) anantibody for the immunosuppressant drug and (II) an enzyme and (ii)magnetic particles comprising the immunosuppressant drug or an analogthereof. The medium is examined for the presence of a complex comprisingthe immunosuppressant drug and the antibody for the immunosuppressantdrug, the presence and/or amount of the complex indicating the presenceand/or amount of the immunosuppressant drug in the sample.

Another embodiment of the present invention is a method for determiningan immunosuppressant drug in a sample suspected of containing animmunosuppressant drug. A combination is formed in a medium wherein thecombination comprises the sample, a releasing agent for releasing theimmunosuppressant drug and its metabolites from endogenous bindingmoieties and a selective solubility agent for the immunosuppressant drugand its metabolites. The selective solubility agent comprises a watermiscible, non-volatile organic solvent and the concentration of theselective solubility agent in the medium is sufficient to provide forsubstantially equal solubility of the immunosuppressant drug and itsmetabolites in the medium. The medium is incubated under conditions toprovide for substantially equal solubility of the immunosuppressant drugand its metabolites in the medium. To the medium is added (i) aphotosensitizer associated with a first particle and being capable ofgenerating singlet oxygen, and (ii) a chemiluminescent compositionactivatable by singlet oxygen and associated with a second particle,wherein an antibody for the immunosuppressant drug is associated withthe first particle or the second particle or both. The combination issubjected to conditions for binding of the antibody to theimmunosuppressant drug, if present. The photosensitizer is irradiatedwith light and the amount of luminescence generated by thechemiluminescent composition is detected. The amount of luminescence isrelated to the amount of the immunosuppressant drug in the sample.

Alternatively, in the above embodiment, one of the first particle or thesecond particle comprises the antibody and the other particle comprisesa drug analog for the immunosuppressant drug. The combination issubjected to conditions for competition of the drug analog coatedparticles and the immunosuppressant drug, if present, to the antibodyfor the drug. Alternatively, in the above embodiment, the first particleor the second particle comprises streptavidin, which combines with abiotinylated analog for the immunosuppressant drug in the medium. Thecombination is subjected to conditions for competition of biotinylateddrug analog and the immunosuppressant drug for the antibody for thedrug. In either of the above alternative embodiments, thephotosensitizer is irradiated with light and the amount of luminescencegenerated by the chemiluminescent composition is detected. The amount ofluminescence is related to the amount of the immunosuppressant drug inthe sample.

The following examples further describe the specific embodiments of theinvention by way of illustration and not limitation and are intended todescribe and not to limit the scope of the invention. Parts andpercentages disclosed herein are by volume unless otherwise indicated.

EXAMPLES Materials

All chemicals were purchased from the Sigma-Aldrich Company (St. LouisMo.) unless otherwise noted. Sirolimus powder and its metabolites exceptfor 27,39-O-didesmethyl (or 32,41-O-didesmethyl) sirolimus were allobtained from Wyeth Pharmaceuticals. 27,39-O-didesmethyl (or32,41-O-didesmethyl) sirolimus was obtained from Dr. Uwe Christainslaboratory at Department of Anesthesiology, University of ColoradoHealth Sciences Center, Denver, Colo.

Testing was done using the DIMENSION® RxL analyzer, available from DadeBehring Inc., Newark Del. The instrument was employed using ACMIAimmunoassay technology. The ACMIA assay method is disclosed in U.S. Pat.Nos. 7,186,518, 5,147,529, 5,128,103, 5,158,871, 4,661,408, 5,151,348,5,302,532, 5,422,284, 5,447,870, 5,434,051, the disclosures of which areincorporated herein in their entirety). In the embodiment of the ACMIAmethod used herein and discussed in more detail below, competitionbetween sirolimus analog on chrome particles and sirolimus (SIRO) inpatient samples for antibody for sirolimus conjugated to an enzyme(conjugate) was utilized to determine the amount of sirolimus in thepatient samples. Conjugate that binds to the sirolimus analog on chromeparticles was removed by magnetic separation. The enzymatic activityfrom conjugate remaining in the supernatant is measured and is directlyproportional to the amount of sirolimus in the patient sample. In theACMIA assay format employed, the enzymatic activity observed whentesting a sample containing no sirolimus was indicative of the amount ofenzymatic activity that was not bound to active antibody (i.e., cannotbind sirolimus on chrome particles). The enzymatic activity observedwhen no chrome particle is present is indicative of the total amount ofenzymatic activity in the conjugate. These values can be used toestimate the percent of enzymatic activity bound to active antibody.

Example 1 Automated Immunoassay for Hydrophobic Drugs with VaryingDegrees of Metabolite Cross-Reactivity Utilizing a Non-ManualPretreatment

Preparation of Pretreatment Solution without FK-506 Carbamate (FKE)

This pretreatment solution was prepared to contain 6.8 mg/mL PIPES™ 1.5sodium salt, 0.3 mg/mL EDTA Disodium, 1.0 mg/mL Saponin, 0.09% PLURONIC®25R2, 0.2% Proclin 300, 0.024 mg/mL Neomycin sulfate and 0.99 mg/mLNaN₃, pH 6.5. No organic solvent was added in this solution.

Preparation of Pretreatment Solution Containing 3.75 μg/mL FKE

This pretreatment solution was prepared to contain 3.75 μg/mL of aFK-506 carbamate compound (or tacrolimus ester), 6.8 mg/mL PIPES™ 1.5sodium salt, 0.3 mg/mL EDTA Disodium, 1.0 mg/mL Saponin, 0.09% PLURONIC®25R2, 0.2% Proclin 300, 0.024 mg/mL Neomycin sulfate and 0.99 mg/mLNaN₃, pH 6.5. No organic solvent was added in this solution. The FKEconcentration in the final reaction mixture was 0.86 μg/mL.

Preparation of Pretreatment Solution Containing 15 μg/mL FKE

This pretreatment solution was prepared to contain 15 μg/mL of a FK-506carbamate compound (or tacrolimus ester), 6.8 mg/mL PIPES™ 1.5 sodiumsalt, 0.3 mg/mL EDTA Disodium, 1.0 mg/mL Saponin, 0.09% PLURONIC® 25R2,0.2% Proclin 300, 0.024 mg/mL Neomycin sulfate and 0.99 mg/mL NaN₃, pH6.5. No organic solvent was added in this solution. The FKEconcentration in the final reaction mixture was 3.4 μg/mL.

Preparation of Pretreatment Solution Containing 10% Dimethyl Sulfoxide(DMSO)

This pretreatment solution was prepared by adding DMSO to a finalconcentration of 10% (v/v) into a buffer containing 6.8 mg/mL PIPES™ 1.5sodium salt, 0.3 mg/mL EDTA Disodium, 1.0 mg/mL Saponin, 15 μg/mL of aFK-506 carbamate compound (or tacrolimus ester), 0.09% PLURONIC® 25R2,0.2% Proclin 300, 0.024 mg/mL Neomycin sulfate and 0.99 mg/mL NaN₃, pH6.5. The concentration of DMSO in the final reaction mixture wasapproximately 2.3%.

Preparation of Pretreatment Solution Containing 15% DMSO

This pretreatment solution was prepared by adding DMSO to a finalconcentration of 15% (v/v) into a buffer containing 6.8 mg/mL PIPES™ 1.5sodium salt, 0.3 mg/mL EDTA Disodium, 1.0 mg/mL Saponin, 15 μg/mL of aFK-506 carbamate compound (or tacrolimus ester), 0.09% PLURONIC® 25R2,0.2% Proclin 300, 0.024 mg/mL Neomycin sulfate and 0.99 mg/mL NaN₃, pH6.5. The concentration of DMSO in the final reaction mixture wasapproximately 3.4%.

Preparation of Pretreatment Solution Containing 10% 1-Methoxy-2-propanol

This pretreatment base solution was prepared by adding1-methoxy-2-propanol (MP) to a final concentration of 10% (v/v) into abuffer containing 6.8 mg/mL PIPES 1.5 sodium salt, 0.3 mg/mL EDTADisodium, 1.0 mg/mL Saponin, 15 μg/mL of a FK-506 carbamate compound (ortacrolimus ester), 0.2% Proclin 300, 0.024 mg/mL Neomycin sulfate and0.99 mg/mL NaN3, pH 6.5. The concentration of MP in the final ACMIAreaction mixture was approximately 2.3%.

Table 1 shows the composition of the pretreatment reagent for use inpretreating a sample containing sirolimus (AI=as indicated).

TABLE 1 Composition of the pretreatment reagent for the sirolimus ACMIAassay Name Qty. (per mL) Function FK506 Ester 15 μg dissociatesSirolimus from binding protein DMSO or MP AI dissolve drug andmetabolites SesquiNa PIPES 6.8 mg buffer EDTA Disodium 0.3 mg preventingclot-formation Saponin 1.0 mg blood cell lysis Pluronic 0.9 μL Proclin300 2 μL preservative Neomycin Sulfate 0.024 mg preservative NaN3 0.99mg preservative, matrixPreparation of Anti-Tacrolimus Antibody-β-Galactosidase Conjugate

Monoclonal anti-sirolimus antibody (Wyeth Pharmaceuticals, CambridgeMass.) was conjugated to β-galactosidase using a standardheterobifunctional SMCC (succinimidyltrans-4-(N-maleimidylmethyl)cyclohexane-1-carboxylate) linker accordingto known techniques. The antibody conjugate solution containedapproximately 7.5 μg/mL anti-sirolimus antibody-β-galactosidaseconjugate, 30 mg/mL protease free bovine serum albumin, 0.126 mg/mLMgCl₂, 0.03 mL/mL of Ethylene glycol, 35.14 mg/mL PIPES 1.5 sodium salt,50 mg/mL NaCl and beta-gal mutein (inactivated beta-galactosidase), pH6.5.

Magnetic Chrome Particle Preparation

Sirolimus chrome particles (immunoassay solid phase) were prepared byconjugating sirolimus-C26- or -C32-CMO conjugate to DA10-Dexal-ChromiumDioxide particles using N-hydroxysuccinimide (NHS) ester and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) chemistry. See, forexample, U.S. Pat. No. 6,231,982, the relevant disclosure of which isincorporated herein by reference. The sirolimus chrome particles arethen made into sirolimus chrome particle tablets. Each sirolimus tabletcontains approximately 2 mg sirolimus chrome particle slurry, 30.4 mgtrehalose dihydrate and 3.6 mg CARBOWAX® 100 μm.

Sirolimus Assay

The principle and operation of the ACMIA assay for sirolimus were asfollows: pretreatment reagent without FKE or with FKE containing organicsolvent as the selective solubility agent was added to a reaction vesselon the DIMENSION® RxL analyzer. Next, 18 μL of whole blood containingsirolimus or its metabolites was added. The whole blood was sampled froma standard cup by first mixing the blood with the ultrasonic sampleprobe. The mixing of whole blood sample with the pretreatment solutionensured the hemolysis of the whole blood and the displacement of theprotein bound sirolimus molecules from their binding sites when thetacrolimus carbamate molecules were present. Anti-sirolimusantibody-β-galactosidase conjugate (50 μL) was added next and wasallowed to react with sirolimus in the sample. The chrome particles withimmobilized sirolimus-CMO-DA10-Dexal were added (50 μL) and were allowedto bind the un-bound conjugate. The sirolimus bound anti-sirolimusantibody-β-galactosidase conjugate did not bind to the chrome particlesbut remained in the supernatant when a magnetic field was applied to theabove reaction mixture to separate the solution from the chromeparticles. The sirolimus-bound conjugate was detected by transferringthe supernatant from the reaction vessel to a photometric cuvette andmeasuring the enzymatic rate of the conjugate in the presence ofchlorophenol red-β-D-galactopyranoside (CPRG). The rate was measuredbichromatically at 577 and 700 nm.

Comparison of Different Pretreatment Reagents

DMSO at 10 or 15% or MP at 10% with FKE were used to make separatepretreatment solutions (as discussed in detail above) for the ACMIAassay conducted on the DIMENSION® RxL analyzer for measuring sirolimusand its metabolites concentrations in whole blood samples. Anotherpretreatment solution was made without the above-mentioned organicsolvents and FKE as control for the assay (“Control”). The pretreatmentsolutions spiked with and without the mentioned organic solvents wereused to prepare the reagent cartridges for the sirolimus ACMIA assay onthe DIMENSION® clinical chemistry analyzer. When the above-mentionedsolvents and FKE were not used, all the metabolites showed highrecoveries in the whole blood samples. In the following tables, therecoveries of sirolimus metabolites are reported as the percent of therecovery of the parent drug, sirolimus.

FIG. 1 shows that a substantial amount of sirolimus drug was released byFKE as witnessed by a large increase in the signal separation versus theno FKE control. The above experiment was performed with no organicsolvent added and the reaction mixture was basically an aqueoussolution. Addition of water miscible organic solvent such as alcohol orDMSO in the pretreatment reagent significantly reduced the metabolitecross-reactivity as indicated in the table. When no organic solvent wasadded, the metabolite cross-reactivity was the highest (the control inthe Table 2 below). The results are summarized in Table 2.

TABLE 2 Metabolite* cross-reactivity using reagent containing varyingorganic solvent % Solvent in % Solvent in Pretreatment Reaction % Cross-Treatment Rgt** Mixture Reactivity Control — — 180 DMSO 10 2.3 100 DMSO15 3.4 88 Methoxypropanol 10 2.3 92 *27, 39 Didesmethyl Sirolimus isused in this study **The pretreatment reagent contains 15 μg/mL FK506ester

In the study referred to in Table 2 above, 27, 39 didesmethyl sirolimuswas used for the organic solvent screening due to its relatively highhydrophilicity.

Table 3 illustrates the effect of FKE and organic solvent exemplified byDMSO on the percent cross-reactivity of sirolimus metabolites.

TABLE 3 Effect of FKE and DMSO on sirolimus metabolite cross-reactivityPT* with PT with Neither PT with 15 μg/mL FKE Nor 15 μg/mL FKE &Metabolite DMSO FKE 15% DMSO 41-O-demethyl-(south) 62 7 −1 hydroxysirolimus 7-O-demethyl sirolimus 6 7 −3 11-hydroxy sirolimus 315 78 3511-hydroxy sirolimus 114 34 9 (isomer of the above) (south) hydroxysiro-2H 17 11 0 (N-oxide)-hydroxy sirolimus 152 49 13 (south) hydroxysiro-2H (isomer) 32 7 0 41-O-demethyl-(south) dihydroxy siro-2H41-O-demethyl sirolimus 48 39 40 32-O-desmethyl sirolimus 34 8 −3 27,39Didesmethyl sirolimus 569 165 88 *PT = Pretreatment Reagent

In the regular aqueous immunoassay reagents containing neither FKE norDMSO, 315% cross-reactivity for 11-hydroxy sirolimus was detected due toits hydrophilic nature. In the presence of FKE but without DMSO, itscross-reactivity was 78%, significantly lower than that in the absenceof FKE but higher than that when both FKE and DMSO were present. Thecross-reactivity of 11-hydroxy sirolimus was the lowest (35%) when bothFKE and DMSO are formulated in the pretreatment reagent. Thecross-reactivity of other sirolimus metabolites followed the samepattern: cross-reactivity is the highest when the pretreatment reagentcontains neither FKE nor DMSO, and the lowest when both are present.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims. Furthermore, the foregoing description,for purposes of explanation, used specific nomenclature to provide athorough understanding of the invention. However, it will be apparent toone skilled in the art that the specific details are not required inorder to practice the invention. Thus, the foregoing descriptions ofspecific embodiments of the present invention are presented for purposesof illustration and description; they are not intended to be exhaustiveor to limit the invention to the precise forms disclosed. Manymodifications and variations are possible in view of the aboveteachings. The embodiments were chosen and described in order to explainthe principles of the invention and its practical applications and tothereby enable others skilled in the art to utilize the invention.

1. A method for determining a hydrophobic immunosuppressant drug in asample suspected of containing a hydrophobic immunosuppressant drug, themethod comprising: (a) providing in combination in a medium: (i) asample suspected of containing a hydrophobic immunosuppressant drug,(ii) a releasing agent for releasing the hydrophobic immunosuppressantdrug and the metabolites of the immunosuppressant drug from endogenousbinding moieties that bind the immunosuppressant drug and themetabolites, and (iii) a selective solubility agent for the hydrophobicimmunosuppressant drug and the metabolites wherein the selectivesolubility agent comprises a water miscible, non-volatile organicsolvent and wherein the concentration of the selective solubility agentin the medium is sufficient to enhance the bioavailability of thehydrophobic immunosuppressant drug over that of the metabolites in themedium, (b) incubating the medium under conditions for enhancing thebioavailability of the hydrophobic immunosuppressant drug over that ofthe metabolites in the medium, (c) adding to the medium of step b), (i)an enzyme labeled antibody that is specific for and binds to theimmunosuppressant drug and (ii) magnetic particles having bound theretoan analog of the hydrophobic immunosuppressant drug analog thereof, and(d) examining the medium and detecting for the presence of the label,the presence thereof indicating the presence of a complex comprising thehydrophobic immunosuppressant drug and the antibody specific for theimmunosuppressant drug, the presence and/or amount of the complexindicating the presence and/or amount of the hydrophobicimmunosuppressant drug in the sample.
 2. The method according to claim 1wherein the immunosuppressant drug is selected from the group consistingof tacrolimus, cyclosporin, rapamycin and everolimus.
 3. The methodaccording to claim 1 wherein the selective solubility agent is a C₂ toC₆ polyol comprising 2 to 3 hydroxy groups, a C₂ to C₆ sulfoxide, a C₂to C₆ sulfone or a C₂ to C₆ amide, a C₂ to C₆ mono-, di- and tri-etherof a polyol or a C₂ to C₆ mono-, di- and tri-esters of a polyol.
 4. Themethod according to claim 1 wherein the selective solubility agent isethylene glycol, glycerol, 1-methoxy-2-propanol, dimethyl sulfoxide,dimethyl sulfone or dimethylformamide.
 5. The method according to claim1 wherein the releasing agent is an analog of the immunosuppressantdrug.
 6. The method according to claim 1 wherein the examining comprisesseparating the magnetic particles from the medium.
 7. The methodaccording to claim 1 wherein the magnetic particles are chromium dioxideparticles.